1. Field of the Invention
The invention relates to improved control of deleterious sulfate-reducing bacteria which produce corrosion and noxious sulfide ions or sourness in aqueous environments, particularly oil reservoirs, oil and gas wells, pipelines, vessels, cooling towers, and the like.
In one aspect, the invention relates to a process for determining the presence of sulfate-reducing bacteria in an aqueous environment utilizing an immunoassay technique.
In another aspect, the invention relates to apparatus for determining the presence of sulfate-reducing bacteria.
2. Brief Description of the Prior Art
Sulfate-reducing bacteria are strictly anaerobic eubacteria. They have considerable physiological and morphological diversity, and no simple classification scheme is recognized. They are variously classified in at least the following genera: Desulfovibrio, Desulfotomaculum, Desulfomonas, Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema, Desulfosarcina, and Thermodesulfobacterium. Furthermore, morphological or physiological similarities among the sulfate-reducing bacteria do not necessarily reflect close generic relationships. The sulfate-reducing bacteria only have in common their ability to utilize sulfate as a terminal electron donor and the fact that they are all anaerobes. When the term "sulfate-reducing bacteria" is used herein these two criteria apply, regardless of taxonomic classification, even to the extent that microbes other than "bacteria" might be included.
In nature, the sulfate-reducing bacteria exploit an ecological niche wherein anaerobic conditions prevail. The greatest variety of sulfate-reducing bacteria is found in nature in permanently anaerobic sulfate rich sediments of low or moderate temperature and salinities, such as the reduced zone in sediments of estuaries and marine habitats. Reduction of the sulfate ion, ultimately to H.sub.2 S, is employed in lieu of reduction of oxygen. Nearly all compounds known to be degradation products of carbohydrates, proteins, nucleic acids, and lipids are utilized by the sulfate-reducing bacteria. Typical electron donors for sulfate-reducing bacteria are H.sub.2, formate, acetate, propionate, higher straight and branched chain fatty acids, monohydroxy alcohols, lactate, dicarboxylic acids, phenyl-substituted carboxylic acids, and related cyclic compounds. In short, they like an anaerobic aqueous medium having sulfate ions to "breathe" and metabolic leftovers to "eat".
Therein lies the problem.
Many industries, including the oil and gas industry, the chemical industry, the electric utility industry, and several other industries have created almost ideal habitats for the sulfate-reducing bacteria in their operations. For example, sulfate-reducing bacteria can infest an oil reservoir, particularly when a water flood is being conducted, so as to form large quantities of H.sub.2 S and turn the reservoir sour with resulting corrosion, separation, and safety problems. Sulfate-reducing bacteria can inhabit well bores, pipelines and other tubulars, storage vessels, and the like causing production of H.sub.2 S and severe corrosion problems. They may inhabit a biological niche below colonies of other microorganisms in vessels, pipelines, on cooling towers, and elsewhere, and thus form H.sub.2 S and/or other metabolic products that also cause severe corrosion problems.
Though a variety of control measures can be employed to halt or mitigate the deleterious effects of the sulfate-reducing bacteria in such industrial operations, typically, particularly in the oil industry, a bactericidal or bacteriostatic agent is introduced into the system. For example, chlorine, ozone, acrolein, quaternary ammonium salts, peroxides, or a number of other known measures can be employed.
However, in order for control measures to be both effective in controlling sulfate-reducing bacteria infestations, while yet being cost effective and not introducing unnecessary deleterious effects into the system, better methods are needed to determine the presence and/or quantity of sulfate-reducing bacteria present in the environments to be treated.
Consequently, efforts have been made to develop a method for determining sulfate-reducing bacteria in aqueous environments. It is desirable that a means to detect these bacteria be capable of use in the field, be broad yet reliable, sensitive, quantitative, rapid, and simple enough that it can be performed by untrained personnel. An immunological means of detecting essentially all of the sulfate-reducing bacteria, meeting the above criteria is a subject of this invention. It is possible because the genera of sulfate-reducing bacteria contain unique enzyme proteins which nevertheless share an antigenic site.
A detailed discussion of selected relevant prior art references follows:
In Biotechnology News, Aug. 15, 1986, a kit based on a half dozen polyclonal antibody preparations to the surface antigens of different sulfate-reducing bacteria and immunofluorescence assays for the purpose of detecting sulfate-reducing bacteria was disclosed. There are two mixtures of the antibodies, one for marine applications and one for use on land. The kit described above, based on antibodies to surface antigens known to be different among the different genera of sulfate-reducing bacteria, requires mixtures of antibodies and must rely on the assumption that a mixture contains an antibody to every genus and species of sulfate-reducing bacteria likely to be encountered.
A paper by A. D. Smith, Arch. Microbiol. 133: 118-121 (1982), illustrates the possible weakness of the use of surface antigens for the identification of sulfate-reducing bacteria. Five specific antisera were prepared against 5 strains of sulfate-reducing bacteria and one polyvalent antiserum was prepared by mixing equal volumes of the five specific antisera. The sera were tested against 44 strains of the genera Desulfovibrio and Desulfotomaculum along with 4 control organisms. Immunological reactivity was mainly strain specific although weak reactivity was seen both within and between groups. None of the antisera including the polyvalent antiserum successfully detected all the sulfate-reducing bacteria in the test. Cross reactivity with control bacteria was weak or absent.
Norqvist et al., Applied and Environmental Microbiology 50: 31-37 (1985), disclose that the envelope proteins of some strains of Desulfovibrio were quite different and that the envelope proteins of at least one species of Desulfotomaculum was unique from that of several other species. This study illustrates why one would not expect antisera against surface antigens to cross react adequately to detect sulfate-reducing bacteria of different genera. It also points out the diversity of protein envelope molecules among the sulfate-reducing bacteria.
Use of antibodies against surface antigens to detect bacteria has been previously used in detecting other bacteria. For example, a need to be able to quickly and accurately detect the presence of the genus Neisseria (the organism causing gonorrhea) exists. One of the conventional tests uses serological methods. Limitations of the use of serological methods for these bacteria were pointed out in the publication "International Symposium on Gonorrhea", B. B. Diena, Ed., a collection of papers presented at the October, 1973 International Symposium on Gonorrhea sponsored by the Health Protection Branch, Health and Welfare Canada, Ottawa, in the chapter entitled "Uses and Limitations of Serologic Tests for Gonorrhea: An Overview" by L. C. Norins, P 34-43.
To overcome the limitations of detection of Neisseria by antibodies to bacterial surface antigens, H. H. Weetall, U.S. Pat. No. 4,166,765, U.S. Pat. No. 4,188,371 and U.S. Pat. No. 4,245,038, developed several relatively simple and quick tests for the presence of Neisseria in liquid samples founded upon the discovery of an enzyme in Neisseria bacteria which is specific to the genus Neisseria. An antiserum was prepared against the enzyme tentatively identified as 1,2-propanediol dehydrogenase from Neisseria bacteria and used in an immunoassay to detect the presence of said enzyme in lysates of bacterial samples.
In the present application, it is disclosed that antibodies to a purified preparation of the enzyme adenosine 5'-phosphosulfate reductase (APS reductase) from one genus of sulfate-reducing bacteria surprisingly cross-reacts with molecules in lysates of the bacteria of other genera of sulfate-reducing bacteria.
As described by Widdel, in Anaerobic Bacteria in Habitats Other Than Man, 1986, eds. E. M. Barnes, and G. C. Mead, Blackwell Scientific, the sulfate-reducing bacteria consist of 8 genera. They are diverse both physiologically and morphologically, and the guanine plus cytosine content (a measure of closeness of composition of their DNA's) ranges widely, (from 34 to 67 mol %).
H. D. Peck, Chapter 18, pages 309-335, in Microbial Chemoautotrophy, edited by W. R. Strohl and O. H. Tuovinen, published by the Ohio State University Press, Columbus, Ohio discloses the physiological diversity of the sulfate-reducing bacteria.
Singleton, et al., Arch. Microbiol. 139: 91-94, (1984), using an indirect enzyme-linked immunosorption assay for cytochrome c.sub.3 to study the immunological relatedness of cytochromes c.sub.3 of different sulfate-reducing bacteria showed that cytochromes c.sub.3 from various strains of the genus Desulfovibrio contain markedly different antigenic determinants. The authors concluded that the diversity of the sulfate-reducing bacteria may be much greater than has been previously supposed.
Skyring et al., Can. J. Microbiol., 19: 375-380 (1973), point out that the dissimilatory sulfate-reducing bacteria are so called because of their unique energy metabolism which is linked to the reduction of sulfate to hydrogen sulfide. They suggest that all of the sulfate-reducing bacteria may reduce sulfate by means of a common mechanism. They state that common biochemical ancestry may be reflected in similarities of enzymes of the sulfate reduction pathway more than of other cellular constituents. They compared the electrophoretic properties of three enzymes of the sulfate reduction pathway including adenosine 5'-phosphosulphate reductase (APS reductase) from 13 strains of dissimilatory sulfate-reducing bacteria from 2 genera. They found a similarity in the electrophoretic behavior of the APS reductase from some of the strains.
Stille et al. Arch. Microbiol. 137: 140-150 (1984) disclose a comparison of properties of APS reductases purified at the time of the study. The enzymes differ as they range in molecular weight from 1.7.times.10.sup.5 to 2.2.times.10.sup.5 and contain from 4 to 8 non heme iron atoms per molecule of enzyme. Other similarities and differences are disclosed.
Aketagawa et al. J. Gen. Appl. Microbiol. 31: 347-357 (1985) determined the immunological cross-reactivities among sulfite reductase, hydrogenases, and somatic antigens of 10 strains in 5 species of the single genus Desulfovibrio. They disclose that the sulfite reductases from Desulfovibrio share common antigenic determinants. However, the hydrogenases, which had different physico-chemical properties from strain to strain showed limited immunological cross-reactivities. The immunological cross-reactivities along with other known features of the sulfite reductases of Desulfovibrio suggest that the structure of the sulfite reductase has been conserved better than other cellular components during evolution. There was little immunological cross-reactivity among the hydrogenases from the 10 strains and in agreement with the previously cited results, none of the antisera against somatic antigens tested reacted with all the 10 species within the genus though there was cross-reactivity among some of the strains.
Despite all the heterogeneity of antigens and intracellular molecules of the sulfate-reducing bacteria as disclosed above, the present invention makes use of the findings that APS reductases in the sulfate-reducing bacteria share sufficient immunological cross-reacting sites to permit their detection by immunological means and thereby to serve as a means of detecting the presence of sulfate-reducing bacteria in lysed samples. Even though APS reductases are known to exist in some sulfide oxidizing bacteria, they do not have sufficient immunological cross-reactivity with those from sulfate-reducing bacteria to interfere with the detection of sulfate-reducing bacteria by the means of this invention.
Accordingly, the invention provides an improved method for controlling sulfate reducing bacteria in industrial operations, particularly in the oil industry. It provides apparatus for rapidly determining the presence and/or quantity of sulfate-reducing bacteria in an aqueous environment. It provides an accurate, easily conducted, and rapid method of detecting and/or quantifying sulfate-reducing bacteria in industrial systems, or elsewhere.